Thin collagen tissue for medical device applications

ABSTRACT

This invention relates to processes of preparing heterogeneous graft material from animal tissue. Specifically, the invention relates to the preparation of animal tissue, in which the tissue is cleaned and chemically cross-linked using both vaporized and liquid cross-linking agents, resulting in improved physical properties such as thin tissue and lowered antigenicity, thereby increasing the ease of delivering the tissue during surgery and decreasing the risk of post-surgical complication, respectively.

CROSS REFERENCE TO RELATED APPLICATIONS

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STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

No federal government funds were used in researching or developing thisinvention.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

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REFERENCE TO A SEQUENCE LISTING

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BACKGROUND

1. Field of the Invention

This invention relates to bioprosthetic transcatheter valve and implantmaterial, processes for preparing bioprosthetic transcatheter valve andimplant material from animal tissue, and methods of use thereof.Specifically, the invention relates to the preparation of animal tissue,in which the tissue is cleaned, chemically cross-linked using bothvaporized and liquid cross-linking agents, and compressed, resulting inan improved bioprosthetic or implantable material that is substantiallynon-antigenic, non-thrombogenic, resistant to calcification, durable,and thin enough to be used in applications requiring extremely smallvalves or implants.

2. Background of the Invention

The use of prepared heterogenous graft material for human surgicalimplantation is well known. More specifically, the use of treated animaltissue as human tissue grafts, replacement valves, and similarimplantation surgical procedures is well known. However, problems ofimmunogenicity, thrombogenicity, calcification, material strength, andsize have not been adequately addressed in the prior art.

Prior to the present invention, animal tissue specimens for surgical usewere prepared by first harvesting the selected tissue from beef cattleor other meat supplying animals at the slaughter house. The harvestedtissues were then transported to a laboratory where the material wascleaned by mechanically stripping away fat tissue and other undesiredcomponents from the harvested specimen material. Next, the cleanedtissue specimen was subjected to a “wet” cross-linking operation inwhich it is soaked for a predetermined time in a glutaraldehyde solutionand finally was dehydrated in an alcohol solution. Subsequently, thesample was thoroughly rinsed to remove traces of the ethyl alcohol andglutaraldehyde and then was packaged in a vial containing a one percentpropylene oxide solution as a sterilant.

While the use of cattle or other meat supplying animals ensures anadequate supply of tissue for processing, a combination of (i) the lowernatural collagen levels and higher non-collagenous protein levels in thetissue of older animals, (ii) the lack of a processing step toeffectively remove non-collagenous proteins, and (iii) the limitationsof “wet” cross-linking, when used alone, to bond glutaraldehyde withcollagen molecules, results in a product that still exhibits traits ofantigenicity, thrombogenicity and calcification that can result inpost-surgical complications, as well as limited endothelializationproperties.

More specifically, the use of glutaraldehyde alone in chemicalcross-linking of tissue results in a tissue sample wherein the releaseof glutaraldehyde after implantation of the sample results in anincreased risk of inflammation in and around the implanted tissue.

For example, U.S. Pat. No. 6,468,313 to Bio-Vascular, Inc. discloses animplant material in the form of a natural animal tissue cross-linkedinto a pre-formed shape, the tissue being adapted to substantiallyretain its shape when implanted into a body.

In another example, U.S. Pat. No. 5,507,810 to Osteotech, Inc. disclosesfibrous connective tissue for surgical implantation is madesubstantially antigen-free by contact with one or more extractionagents.

In another example, U.S. Pat. No. 4,681,588 to Ketharanathan disclosesmaterial for use in a biological environment is produced by subjecting asheet of parietal pleura to glutaraldehyde tanning.

In another example, U.S. Pat. No. 4,399,123 to Oliver discloses afibrous tissue preparation suitable for homo or heterotransplantationobtained by treating mammalian fibrous tissue with a proteolytic enzymefollowed, if desired, by further treatment with a carbohydrate splittingenzyme.

However, known procedures for treating animal tissue typically result intissue thickness too large for surgical use in applications requiring asmaller valve or implant. Tissue samples of this thickness can limit theuse of smaller gauge catheters in delivering the tissue sample to thearea of the human body in which surgery is to be performed, or limit thetypes of patients that may be treated to large patients only.

For example, bovine pericardial tissue used in the products Duraguard®,Peri-Guard®, and Vascu-Guard®, all products currently used in surgicalprocedures, are marketed as being harvested generally from cattle lessthan 30 months old. However, pericardial tissue from older animals isthicker than younger animals, and thus limits the thinness that can beachieved. Other patents and publications that are directed to thesurgical use of harvested, biocompatible animal tissues may disclosethin tissues, however, these tissues are used only as biocompatible“jackets” or sleeves for implantable stents. Accordingly, these tissuesdo not have the biomechanical, e.g. strength and durability, necessaryfor the construction of bioprosthetic transcatheter valves, or implants.For example, U.S. Pat. No. 5,554,185 to Block discloses an inflatableprosthetic cardiovascular valve which is constructed so as to beinitially deployable in a deflated “collapsed” configuration wherein thevalve may be passed through the lumen of a cardiovascular catheter andsubsequently inflated to an “operative” configuration so as to performits intended valving function at its intended site of implantationwithin the cardiovascular system. In another example, U.S. Pat. No.7,108,717 to Design & Performance-Cyprus Limited discloses a coveredstent assembly comprising a tubular, expandable stent having a metallicframework covered with a cylinder of biocompatible, non-thrombogenicexpandable material, such as heterologous tissue. In another example,U.S. Pat. No. 6,440,164 to Scimed Life Systems, Inc. discloses aprosthetic valve for implantation within a fluid conducting lumen withina body includes an elongate generally cylindrical radially collapsiblevalve body scaffold defining a fluid passageway therethrough forretentive positioning within the lumen. However, these patents describenecessarily elastic materials that are used for covering expandablewire-mesh stents.

Methods do currently exist for production of synthetic bioprostheticmaterials in the form of an acellular collagen-based tissue matrix.However, the product suffers from a strength deficiency, is subject totearing and is not ideal for suture retention. For example, U.S. Pat.No. 5,336,616 to LifeCell Corporation discloses a method for processingand preserving an acellular collagen-based tissue matrix fortransplantation. However, to date, the molecular reasons why naturallymatured collagen is superior to synthetic have not been fullyelucidated.

Accordingly, procedures and devices which address these and otherconcerns are needed in the field.

BRIEF SUMMARY OF THE INVENTION

In a preferred embodiment, there is provided a process of preparing abioprosthetic or implant tissue material for use in surgical procedureson humans comprising the steps of: (a) vapor cross-linking apre-digested compressed tissue specimen by exposing the tissue specimento a vapor of a cross-linking agent selected from the group consistingof aldehydes, epoxides, isocyanates, carbodiimides, isothiocyanates,glycidalethers, and acyl azides; and (b) chemically cross-linking thevapor-cross-linked tissue specimen by exposing the vapor-crosslinkedtissue specimen to an aqueous crosslinking bath for a predeterminedtime, such crosslinking bath containing a liquid phase of a crosslinkingagent selected from the group consisting of aldehydes, epoxides,isocyanates, carbodiimides, isothiocyanates, glycidalethers, and acylazides.

In a preferred embodiment, there is also provided a tissue materialprepared according to the process herein. In another preferredembodiment, the tissue specimen is harvested from a porcine, ovine orbovine animal. Alternatively, the predetermined tissue specimen is takenfrom a bovine animal 30 days old or less. In one preferred embodiment,the tissue specimen is taken from an animal that is not more than about10 days old, and in a preferred embodiment about 5 days old.

In another preferred embodiment the harvested tissue specimen comprisesa collagen-based tissue selected from the group consisting ofpericardium, dura mater, heart valves, blood vessels, fascia, ligaments,tendons, and pleura tissue.

In preferred processes, the tissue specimen is subjected to chemicaldehydration/compression and mechanical compression before cross-linking,and/or the pre-digested tissue specimen is provided by digesting aharvested, cleaned pericardial tissue in a solution containing asurfactant. In a preferred embodiment, the surfactant is 1% sodiumlaurel sulfate.

Preferably, the chemical dehydration/compression comprises subjectingthe tissue specimen to hyperosmotic salt solution.

The mechanical compression may preferably comprise subjecting the tissuespecimen to a roller apparatus capable of compressing the tissuespecimen to a thickness ranging from about 0.003′ (0.0762 mm) to about0.010″ (0.254 mm).

In another preferred embodiment, there is provided a process ofpreparing animal-derived collagen tissue material for use in surgicalprocedures on humans comprising the steps of: (a) vapor cross-linking apre-digested collagen tissue specimen by exposing the tissue specimen toa formaldehyde vapor phase; and (b) subjecting the vapor-crosslinkedcollagen tissue specimen to an aqueous glutaraldehyde bath for apredetermined time.

Also contemplated is a tissue for bioprosthetic or implant use in thehuman body prepared according to the process herein.

In another preferred embodiment, there is provided a process ofpreparing a bioprosthetic transcatheter valve material for use insurgical procedures on humans comprising the steps of: (a) vaporcross-linking a pre-digested compressed bovine pericardium tissuespecimen by exposing the tissue specimen to a formaldehyde vapor phase;and (b) subjecting the vapor-crosslinked tissue specimen to an aqueousglutaraldehyde bath for a predetermined time.

In another preferred embodiment, it is contemplated to include a step ofsterilizing the cross-linked tissue specimen.

In another preferred embodiment, it is contemplated to further comprisewherein the compression of the tissue specimen is subjecting to chemicaldehydration/compression and mechanical compression.

In another preferred embodiment, it is contemplated to further comprisewherein the pre-digested tissue specimen is provided by digesting aharvested, cleaned bovine pericardial tissue in a solution containing asurfactant. Preferably, the surfactant is 1% sodium laurel sulfate.

In another preferred embodiment, it is contemplated to further comprisewherein the chemical dehydration/compression comprises subjecting thetissue specimen to hyperosmotic salt solution and wherein the mechanicalcompression comprises subjecting the tissue specimen to a rollerapparatus capable of compressing the tissue specimen to a thicknessranging from about 0.003′ (0.0762 mm) to about 0.010″ (0.254 mm).

In another preferred embodiment, there is provided a bioprosthetictranscatheter valve material for use in the human body preparedaccording to the processes herein.

In yet another preferred embodiment, there is provided a process ofpreparing heterogenous or homogenous tissue material for use in surgicalprocedures on humans wherein an animal collagen tissue specimen ischemically cross-linked first by exposing the tissue to formaldehydevapor for approximately 10 minutes, and second by immersing the tissuein a glutaraldehyde solution for two consecutive sessions ofapproximately 24 hours each.

In another preferred embodiment, there is provided a process ofconverting pericardial tissue specimen taken from a bovine animal notmore than 30 days old to a non-antigenic, non-thrombogenic,calcification-resistant implantable material for use in surgicalprocedures on humans wherein the pericardial tissue specimen is cleaned,digested by surfactant, compressed to approximately 0.003″ in thickness,vapor cross-linked by exposing the tissue to a vapor-phase cross-linkingagent selected from the group consisting of aldehydes, epoxides,isocyanates, carbodiimides, isothiocyanates, glycidalethers, and acylazides, and liquid-phase cross-linked by immersing the tissue in aliquid cross-linking agent selected from the group consisting ofaldehydes, epoxides, isocyanates, carbodiimides, isothiocyanates,glycidalethers, and acyl azides.

In another preferred embodiment, it is contemplated to provide animplantable tissue material prepared according to the processes herein.

In another preferred embodiment, there is provided a process ofconverting pericardial tissue specimen taken from a bovine animal notmore than 30 days old to a non-antigenic, non-thrombogenic,calcification-resistant implantable material for use in surgicalprocedures on humans wherein the pericardial tissue specimen is cleaned,digested by surfactant, compressed to approximately 0.003″ in thickness,vapor cross-linked by exposing the tissue to formaldehyde vapor forapproximately 10 minutes, and further cross-linked by immersing in aglutaraldehyde solution for at least 24 hours.

In another preferred embodiment, it is contemplated to provide abioprosthetic or implantable material prepared according to the processherein.

Also contemplated is a tissue material as claimed herein, in dehydratedstate for dry packaging.

In another preferred embodiment, it is contemplated to provide a tissuematerial as claimed for use wherein such material is trimmed and/orconfigured to an appropriate shape as replacement tissue for any of thefollowing surgical purposes: stented or stentless pericardial valvereplacement, stented or stentless pulmonic valve replacement,transcatheter valvulare prosthesis, aortic bioprosthesis/valvereplacement or repair, annuloplasty rings, bariatric surgery, duralpatching, enucleation wraps, gastric banding, herniation repair, lungsurgery e.g. lung volume reduction, peripheral arterial or venous valvereplacement, pericardial patching, rotator cuff repair, uretheralslings, valve repair, vascular patching, valve conduit insertion, orarterial conduit insertion.

Preferably, the tissue material as claimed in any of claims comprises avery thin, durable material that ranges from about 0.002″ (0.0508 mm) toabout 0.020″ (0.508 mm) as the average cross-sectional thickness. At thelower end of this range, the focus is on being able to create materials,tissues, and devices that can access applications where a very thin,very durable material is needed. At the upper range of the presentinvention, the focus may not necessarily be on the thinness of thematerial compared to the 0.002″ (0.0508 mm) materials; however, thedurability, and the ability to form materials, tissues and devices froma wider range of starting materials and sources. In another preferredembodiment, the present invention ranges from about 0.002″ (0.0508 mm)and about 0.010″ (0.254 mm) in thickness. In another preferredembodiment, the present invention ranges from about 0.002″ (0.0508 mm)and about 0.005″ (0.127 mm) in thickness. In one preferred embodiment,the present invention averages approximately 0.003″ (0.0762 mm) inthickness.

In another preferred embodiment, it is contemplated to provide a tissuematerial as claimed wherein the tissue material is provided in sterileform and is adapted to be implanted into a body and attached in place.For example, the material may be configured in a spherical form to wrapan orbital implant, or configured to form leaflets in a prosthetictranscatheter valve.

In another preferred embodiment, it is contemplated to provide a stentassembly for maintaining the patency of a body lumen comprising anexpandable stent with or without a biocompatible jacket, and aprosthetic transcatheter valve made from the inventive tissue materialdisposed therein to function as a valve replacement.

It is also contemplated to manufacture a prosthetic transcatheter valvemade from the tissue material configured for delivery within anintravenous catheter measuring 18 or less in french gauge, or evenwithin a gauge 14 or less french gauge.

In a preferred embodiment, a process of preparing animal tissue for usein surgical procedures on humans comprising the steps of:

harvesting a predetermined tissue specimen from a bovine animal at thetime of slaughter of such animal;

cleaning the tissue specimen a first time to remove unwanted components;

digesting the tissue to denucleate and to remove non-collagenousproteins;

cleaning the tissue specimen a second time to remove unwantedcomponents;

chemically compressing the tissue specimen, including with ahyperosmotic solution;

mechanically compressing the tissue specimen;

chemically cross-linking the compressed tissue specimen by exposing thetissue to a vapor selected from the group consisting of aldehydes,epoxides, isocyanates, carbodiimides, isothiocyanates, glycidalethers,and acyl azides, but especially formaldehyde;

chemically cross-linking the compressed tissue specimen by exposing thetissue to an aqueous bath for a predetermined time, such bath containinga solute selected from the group consisting of aldehydes, epoxides,isocyanates, carbodiimides, isothiocyanates, glycidalethers, and acylazides, but especially glutaraldehyde;

sterilizing the tissue specimen, for example, by exposing the tissue toan ethanol soak for a predetermined time; and optionally placing thesterilized tissue specimen in a sterilized package.

Preferred embodiments include, wherein the harvested tissue specimencomprises a collagen-based tissue selected from the group consisting ofpericardium, dura mater, heart valves, blood vessels, fascia, ligaments,tendons, and pleura tissue.

Methods of use include using the tissue configured to an appropriateshape as replacement tissue for any of the following surgical purposes:stented or stentless pericardial valve replacement, stented or stentlesspulmonic valve replacement, transcatheter valvulare prosthesis, aorticbioprosthesis/valve replacement or repair, annuloplasty rings, bariatricsurgery, dural patching, enucleation wraps, gastric banding, herniationrepair, lung surgery e.g. lung volume reduction, peripheral arterial orvenous valve replacement, pericardial patching, rotator cuff repair,uretheral slings, valve repair, vascular patching, valve conduitinsertion, or arterial conduit insertion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart evidencing the steps of the process claimedherein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided as an aid to understanding thedetailed description of the present invention.

“Bobby calf” as used herein means a male or a female calf of a dairy cowthat is slaughtered before weaning, usually not more than 30 days frombirth.

“Collagen” is the most abundant protein in all animal tissue, and is theprimary component of connective tissue. Collagen consists of a proteinwith three polypeptide chains, each containing approximately 1000 aminoacids and having at least one strand of repeating amino acide sequenceGly-X-Y, where X and Y can be any amino acid but usually are proline andhydroxyproline, respectively. Collagen assembles into differentsupramolecular structures and has exceptional functional diversity.

Natural collagen sources contemplated as within the scope of the presentinvention include porcine, ovine, or bovine animals 30 days old or less.In one preferred embodiment, the tissue specimen is taken from anporcine, ovine, or bovine animal that is not more than about 10 daysold, and in a preferred embodiment about 5 days old. Preferredembodiments include specific tissues, wherein the harvested tissuespecimen comprises a collagen-based tissue selected from the groupconsisting of pericardium, dura mater, heart valves, blood vessels,fascia, ligaments, tendons, and pleura tissue.

“Cross-links” are bonds that link one polymer chain to another. They canbe covalent bonds or ionic bonds. “Polymer chains” can refer tosynthetic polymers or natural polymers, including proteins such ascollagen. Examples of some common crosslinkers are the dimethylsuberimidate, formaldehyde and glutaraldehyde. Each of thesecrosslinkers induces nucleophilic attack of the amino group of lysineand subsequent covalent bonding via the crosslinker.

“Pyridyl” encompasses a set of functional groups in the pyridinederivative chemical class with the common structure C₅N₁. A pyridylgroup will bond an aldehyde compound to a collagen protein through thecross-linking process.

“Surfactants” are wetting agents that lower the surface tension of aliquid, allowing easier spreading, and lower the interfacial tensionbetween two liquids. The term surfactant is a blend of “surface activeagent”. Surfactants are usuallyorganic compounds that are amphiphilic,meaning they contain both hydrophobic groups (their “tails”) andhydrophilic groups (their “heads”). Therefore, they are soluble in bothorganic solvents and water. Surfactants are also often classified intofour primary groups: anionic, cationic, non-ionic, and zwitterionic(dual charge). A non-limiting preferred surfactant contemplated hereinis sodium laurel sulfate, although various other surfactants known to aperson of ordinary skill in the art are also contemplated as within thescope of the invention.

The use of Bobby calf (BC) pericardial tissue for prostheses provides anumber of benefits over alternative tissue sources. BC animals areprimarily used for the production of veal, meaning that a large andsteady supply of tissue from such animals is available. BC pericardialtissue is known to be extremely thin, typically in the range of 0.005″to 0.007″ (0.1270 mm to 0.1778 mm). Pericardial tissue from such animalsalso has a very high natural collagen content, providing the tissue bothhigh strength and a variety of biocompatibility benefits, including lowantigenicity, thrombogenicity and calcification potential; highendothelialization; high suture retention; and high bursting strengths.As the animal ages, the natural collagen content of its tissuedecreases, and these biocompatibility benefits also decrease.

Increasing the collagen content of a given specimen of animal tissue,and simultaneously decreasing the presence of non-collagenous proteinsin such tissue results in a heightened biocompatibility of treatedtissue samples. The high natural collagen content of BC pericardialtissue makes it an excellent source of tissue for collagen-enhancingtreatment.

Treatment of BC pericardial tissue for use in surgical transplantationshould begin with an isotonic saline wash at room temperature, whereuponthe sample should be split to form a flat sheet and then returned to thesaline solution to await further processing. Formation of a flat sheetof tissue allows for later trimming and manipulation to form shapesspecifically tailored to individual surgeries.

Washing a tissue sample with a surfactant/water solution for a period ofup to 24 hours can result in a 99:1 post-treatment ratio of collagen tonon-collagenous proteins in the tissue. Such a high ratio greatlyenhances the effectiveness of later collagen cross-linking to furtherimprove biocompatibility of the sample.

Thinness of tissue used for surgical implants and grafts provides manybenefits in surgery. The thickness of such material directly affects thesize of any product or device made with such a material, for example aheart valve, arterial valve, or venous valve. Further, the smaller sizeimpacts the ease with which the material may be introduced into thehuman body, through catheterization or otherwise, as well as the ease ofmanipulation of the material after placement. A thinner sample means alower gauge catheter, and easier intravenous or percutaneous insertion,and thus the ability to treat a higher percentage of the patientpopulation requiring such an intervention.

BC pericardial tissue that has been surfactant treated and attained avery high collagen content becomes ideally suited to compression tofurther thin the tissue sample. BC pericardial tissue of the presentinvention may be about at least 95% to about at least 99% collagen. In anon-limiting preferred embodiment, the collagen content is about 99%.Suspension in a hyperosmotic solution for a period of 30 minutes willsubstantially thin the tissue through partial dehydration.

Collagen-enhanced and partially dehydrated BC pericardial tissue may befurther thinned by means of mechanical compression, and the highbursting strength of such tissue will prevent tearing or weakening ofthe tissue in the process. A preferred method of mechanicallycompressing a tissue sample is to place it between two sheets ofpolyethylene film, each larger in surface area than the tissue sample,and covering the entire upper and lower surfaces of the tissue sample,and placing the sample and film into one of (i) a wringer apparatuscomprising upper and lower electrically or manually driven rollers, withthe gap between such rollers set at approximately 0.002″ to 0.020″ usinga feeler gauge, wherein the tissue sample and film are fed through theapparatus, or (ii) a press apparatus comprising upper and lower plates,wherein the plates are compressed via manual or electrically driventurning mechanism until reaching a gap set at approximately 0.002″ to0.020″ using a feeler gauge, wherein the tissue sample and film are fedthrough the apparatus. Additional preferred methods of mechanicalcompression of a tissue sample include subjecting the sample to a vacuumcompression, or applying weighted or compressive force to the sample.

BC pericardial tissue subjected to dehydration compression andmechanical compression as detailed herein is known to attain a tissuethickness of approximately 0.003″ (0.0762 mm), making BC tissue at least40% thinner than bovine tissue currently in use on the surgical market,without compromising the strength of the tissue, and therefore is highlydesirable as a material for surgical implants and grafts. It iscontemplated as within the scope of the invention that tissuethicknesses of about 0.002″ (0.0508 mm) to about 0.007″ (0.1778 mm),without limitation, may be manufactured according to the inventiveprocess.

Once subjected to compression treatment, the BC pericardial tissue isready for collagen cross-linking Cross-linking is a process well knownin the art for improving the biocompatibility of collagen in a piece oftissue prior to surgical implantation. Processes for collagencross-linking currently known in the art have been limited to the“tanning”, or submersion of a tissue sample in a wet bath containing across-linking agent, such as an aldehyde, as a solute.

An ideal primary method for cross-linking collagen in tissue comprisesplacing such tissue onto a pin frame such that the edges are held firmlyin place. The frame and tissue sample are then placed into a chamberequipped with each of an inlet and outlet port for submission to a“vapor cross-linking” process. The inlet port is attached to a stopperedflask comprising each of an inlet and outlet port and containing a bolusof polyoxymethylene, which flask is gently heated as air flow issimultaneously initiated from the flask into the chamber containing thetissue sample, thereby producing formaldehyde vapors which flood thechamber for a period of 10 minutes, after which time such vapors areevacuated from the chamber and the pin frame and tissue sample areremoved intact therefrom.

The use of heated polyoxymethylene to create formaldehyde vapor issuperior to the known method of heating liquid glutaraldehyde, as thelatter decreases the efficiency of the vapor delivery mechanism byreleasing water vapor. Water vapor will swell the tissue material,whereas the use of a formaldehyde vapor results in a relativelyanhydrous cross-linking process. By not allowing excess water duringthis phase of the process, the tissue material maintains its thinprofile. However, gas cross-linking with formaldehyde is limited by thestructural size of formaldehyde and the available of a single aldehydegroup.

After completion of the vapor cross-linking, the tissue material issubjected to a liquid glutaraldehyde bath. Glutaraldehyde provides afurther cross-linking that results in additional cross-links thatformaldehyde cannot achieve. The presence of two aldehyde groups forcross-linking and the ability to be cross link over a distance sinceglutaraldehyde has a three-carbon chain connecting the two carbonylmoieties further strengthens the tissue material. In one non-limitingpreferred embodiment, the pin frame and tissue sample are thentransferred into an aqueous bath containing 1% 0.01M phosphate bufferedglutaraldehyde and 10% isopropyl alcohol at a temperature ofapproximately 40 degrees C., and gently stirred for a period of not lessthan 24 hours, although variations of glutaraldehyde cross-linking arewell known in the art and are considered within the scope of this stepof the present invention.

Although the combination of vapor formaldehyde cross-linking with wetglutaraldehyde cross-linking results in improved stability of thecross-links when compared to a sample subjected to the latter processalone, the combination of formaldehyde vapor and glutaraldehyde liquidappears to provide an additional benefit to the material that resultsfrom the inventive process. The bioprosthetic or implant material of thepresent invention does not exhibit the immunogenic problems known in theart that accompanies glutaraldehyde cross-linked materials. Priorresearch has shown that glutaraldehyde can trigger a strong inflammatoryreaction within a mammalian body, even including anaphylactic reactions.However, the material produced by the present inventive process isnon-antigenic.

It is believed that the use of glutaraldehyde alone in chemicalcross-linking of tissue is known to create cross-linking that issusceptible to opening and releasing glutaraldehyde after implantationof the sample. The result of such degradation of the cross-links is anincreased risk of inflammation in and around the implanted tissue. Incontrast, when pre-treating the sample with vapor formaldehyde via themethod described herein, the formaldehyde acts as a reducing agent,creating cyclic pyridine molecules. The process of creating stable,non-reactive aromatics on the exposed surface of the collagen isbelieved to progress by nucleophilic attack by formaldehyde on thecarbonyl of the glutaraldehyde-linked amine of the lysine, histidine,and/or arginine, improving the stability of the molecular structure ofthe sample and reducing the antigenicity of the sample compared to asample treated with glutaraldehyde alone. A redcued inflammatoryresponse and lower degree of capsule formation provides a distinctadvantage.

After the completion of wet cross-linking, the tissue sample, stillattached to the pin frame, is sterilized by transferring it to anaqueous bath consisting essentially of a 2% buffered glutaraldehydesolution containing 10% isopropyl alcohol, and is soaked therein at 42degrees C. for a period of no less than 24 hours. Upon completion ofsterilization, the tissue sample is removed from the pin frame.

Finally, the tissue sample is packaged for transport in a containertogether with a sterilizing 0.65%, 0.01M phosphate bufferedglutaraldehyde solution, in which solution the tissue sample may eitherfloat freely or be held stationary by attachment to a mylar film.

Upon removal from packaging, the tissue sample may be trimmed, suturedor otherwise manipulated to form the size and shape necessary for anyimplantation surgery for which such tissue would be appropriate.

For example, in one preferred embodiment, the tissue would be trimmed tofit for any necessary vascular or pericardial patching. In anotherpreferred embodiment, the tissue would be sutured to form a cylinder tocover a mesh stent. In another embodiment, the tissue would be cut intoa leaflet shape for prosthetic transplantation into a mitral valve. Inyet another preferred embodiment, a strip of tissue would be cut andsutured into an annular shape for transplantation into a mitral valve.

Methods of use include using the tissue configured to an appropriateshape as replacement tissue for any of the following surgical purposes:stented or stentless pericardial valve replacement, stented or stentlesspulmonic valve replacement, transcatheter valvulare prosthesis, aorticbioprosthesis/valve replacement or repair, annuloplasty rings, bariatricsurgery, dural patching, enucleation wraps, gastric banding, herniationrepair, lung surgery e.g. lung volume reduction, peripheral arterial orvenous valve replacement, pericardial patching, rotator cuff repair,uretheral slings, valve repair, vascular patching, valve conduitinsertion, or arterial conduit insertion.

Referring now to the FIGURES:

FIG. 1 shows a flow chart evidencing the steps and materials used in theBC pericardial tissue treatment process.

Example 1

In this example, the pericardial sac from a bobby calf is washed withisotonic saline at room temperature, held in saline for processing, theninspected for acceptability and split into a flat sheet approximately7×9 cm in dimension. The washed sample is then subjected to “digestion”,in which a detergent extraction to de-nucleate the tissue and removenon-collagenous proteins, preferably using a 1% sodium lauryl sulfatesolution (SDS). The sample is held in this solution and gently stirredfor up to 24 hours. Histological review of the tissue will indicate thatthe tissue has been de-nucleated and that non-collagenous proteins havebeen almost entirely removed. Gross observation of the tissue willindicate a color change from the original tan/white color to pure white.Digestion has the side effect of swelling the tissue.

Following digestion, the swollen tissue is washed again in isotonicsaline until the SDS is removed. This process also reverses some of theswelling from digestion. Next, the tissue is further “chemicallycompressed” by dehydration by placing it in a hyperosmotic NaCl solutionfor a period of 30 minutes, during which time the sample is gentlystirred. Upon removal, the sample is placed between two sheets ofpolyethylene film and subjected to mechanical compression, either usingmechanical rollers, a press, or similar mechanism, resulting in aflattened sample approximately 0.003″ in thickness. The polymer filmsare then removed and the sample is momentarily held on a dry surface.

Next, the compressed tissue is subjected to an initial cross-linkingphase, in which the tissue is first placed onto a pin frame with alledges of the tissue held in place. The tissue/frame is then placed intoa box or similar chamber with an outlet port, and an inlet port. Theinlet port is attached via a tube to an outlet port emanating from anErlenmeyer flask, which flask is also equipped with a stopper and aninlet port, and contains a bolus of polyoxymethylene. The flask isgently heated, drawing air from the inlet port, releasing thecross-linking agent formaldehyde vapor, and pushing the vapor out theoutlet port and into the chamber containing the tissue sample. Thechamber is flooded with vapor for approximately 10 minutes, after whichthe chamber is evacuated of vapor and the tissue/frame is removed.

The tissue/frame is then transferred to an aqueous bath containing 1%,0.01M phosphate buffered glutaraldehyde and 10% isopropyl alcohol for asecond phase of cross-linking The glutaraldehyde solution will have beenprepared according to the teaching of U.S. Pat. No. 7,303,757. The bathwill be maintained at approximately 40 degrees C., and the submergedtissue will be gently stirred therein for no less than 24 hours. At suchtime, the bath solution will be discarded and replaced, and gentlestirring will resume for a second period of no less than 24 hours.

Upon completion of the second cross-linking phase, the tissue is removedfrom the pin frame and sterilized by immersion for a minimum of 24 hoursin a 2% buffered glutaraldehyde solution containing 10% isopropylalcohol, maintained at approximately 42 degrees C. Upon removal fromthis solution, the sterile tissue is packed for shipment into a 0.65%,0.01M phosphate buffered glutaraldehyde solution, in which the samplemay either float freely, or be attached to a mylar film and heldstationary within the container.

Example 2

In this example, pericardial tissue, valve tissue or tendon tissue fromone of a bovine, porcine or ovine animal is washed with isotonic salineat room temperature, held in saline for processing, then inspected foracceptability and split into a flat sheet. The washed sample is thensubjected to “digestion”, in which a detergent extraction to de-nucleatethe tissue and remove non-collagenous proteins, preferably using asolution in which the solute is sodium lauryl sulfate (SDS) or anothersurfactant. The sample is held in this solution and gently stirred formore than 24 hours. Gross observation of the tissue will indicate acolor change from the original tan/white color to pure white, at whichtime the digestion process will be discontinued. Histological review ofthe tissue will indicate that the tissue has been de-nucleated and thatnon-collagenous proteins have been largely removed. Digestion has theside effect of swelling the tissue.

Following digestion, the swollen tissue is washed again in isotonicsaline until the surfactant is removed. This process also reverses someof the swelling from digestion. Next, the tissue is further “chemicallycompressed” by dehydration by placing it in a hyperosmotic solution fora period of 30 minutes, during which time the sample is gently stirred.Upon removal, the sample is placed between two sheets of polyethylenefilm and subjected to mechanical compression, either using rollers,weights, a press, a vacuum or or similar mechanism, resulting in aflattened sample between about 0.003″ (0.0762 mm) and about 0.007″(0.1778 mm) in thickness. The polymer films are then removed and thesample is momentarily held on a dry surface.

Next, the compressed tissue is subjected to an initial cross-linkingphase, in which the tissue is first placed onto a pin frame with alledges of the tissue held in place. The tissue/frame is then placed intoa box or similar chamber with an outlet port, and an inlet port. Theinlet port is attached via a tube to an outlet port emanating from anErlenmeyer flask, which flask is also equipped with a stopper and aninlet port, and contains a bolus of polyoxymethylene. The flask isgently heated, drawing air from the inlet port, releasing thecross-linking agent formaldehyde vapor, and pushing the vapor out theoutlet port and into the chamber containing the tissue sample. Thechamber is flooded with vapor for approximately 10 minutes, after whichthe chamber is evacuated of vapor and the tissue/frame is removed.

The tissue/frame is then transferred to an aqueous bath containingbetween 0.1%-5.0%, 0.01M phosphate buffered glutaraldehyde and 10%isopropyl alcohol for a second phase of cross-linking. Theglutaraldehyde solution will have been prepared according to theteaching of U.S. Pat. No. 7,303,757, or any functionally similar methodknown to persons of ordinary skill in the art. The bath will bemaintained at approximately 40 degrees C., and the submerged tissue willbe gently stirred therein for no less than 24 hours. At such time, thebath solution will be discarded and replaced, and gentle stirring willresume for a second period of no less than 24 hours.

Upon completion of the second cross-linking phase, the tissue is removedfrom the pin frame and sterilized by immersion for a minimum of 24 hoursin a solution containing between 1%-5% buffered glutaraldehyde and 10%isopropyl alcohol, maintained at approximately 42 degrees C. Uponremoval from this solution, the sterile tissue is packed for shipmentinto a 0.65%, 0.01M phosphate buffered glutaraldehyde solution, in whichthe sample may either float freely, or be attached to a mylar film andheld stationary within the container.

The references recited herein are incorporated herein in their entirety,particularly as they relate to teaching the level of ordinary skill inthis art and for any disclosure necessary for the commoner understandingof the subject matter of the claimed invention. It will be clear to aperson of ordinary skill in the art that the above embodiments may bealtered or that insubstantial changes may be made without departing fromthe scope of the invention. Accordingly, the scope of the invention isdetermined by the scope of the following claims and their equitableEquivalents.

What is claimed is:
 1. A prosthetic tissue material for surgicalpurposes which is substantially non-antigenic, non-thrombogenic,calcification-resistant, and tear-resistant comprising avapor-crosslinked and liquid-crosslinked collagen material comprised of95-99% collagen that is derived from denucleated, digested, andcompressed collagen-based tissue, said prosthetic tissue material havingan average cross-sectional thickness ranging from 0.0508 mm to 0.508 mm.2. The prosthetic tissue material according to claim 1, wherein thetissue material is in dehydrated state for dry packaging.
 3. Theprosthetic tissue material according to claim 1, wherein the tissuematerial is trimmed and/or configured to an appropriate shape as aprosthetic tissue selected from the group consisting of: a stented orstentless pericardial valve replacement, a stented or stentless pulmonicvalve replacement, a transcatheter valve prosthesis, an aorticbioprosthesis/valve replacement or repair, an annuloplasty ring, aprosthesis for bariatric surgery, a dural patch, an enucleation wrap, agastric band, a prosthesis for herniation repair, a prosthesis for lungsurgery e.g. lung volume reduction, a peripheral arterial or venousvalve replacement, a pericardial patch, a prosthesis for rotator cuffrepair, a urethral sling, a prosthesis for valve repair, a vascularpatch, a valve conduit insertion, and an arterial conduit insertion. 4.The prosthetic tissue material according to claim 1, wherein the tissuematerial is between about 0.002″ (0.0508 mm) and about 0.005″ (0.127 mm)in thickness.
 5. The prosthetic tissue material according to claim 1,wherein the tissue material is approximately 0.003″ (0.0762 mm) inthickness.
 6. The prosthetic tissue material according to claim 1,wherein the tissue material is provided in sterile packaging.
 7. Theprosthetic tissue material according to claim 1, wherein the tissuematerial is configured in a spherical form to wrap an orbital implant.8. The prosthetic tissue material according to claim 1, wherein thetissue material is configured to form leaflets in a prosthetictranscatheter valve.
 9. The prosthetic tissue material according toclaim 1, wherein the tissue material is configured as a valvereplacement in an expandable stent assembly for maintaining the patencyof a body lumen.
 10. The prosthetic tissue material according to claim1, wherein the tissue material is configured as a biocompatible jacketfor an expandable prosthetic stent.
 11. The prosthetic tissue materialaccording to claim 1, wherein the prosthetic tissue material isvapor-crosslinked using a vapor-phase cross-linking agent selected fromthe group consisting of aldehydes, epoxides, isocyanates, carbodiimides,isothiocyanates, glycidalethers, and acyl azides.
 12. The prosthetictissue material according to claim 1, wherein the prosthetic tissuematerial is vapor-crosslinked using vapor-phase formaldehyde.
 13. Theprosthetic tissue material according to claim 1, wherein the prosthetictissue material is liquid-crosslinked using a liquid-phase cross-linkingagent selected from the group consisting of aldehydes, epoxides,isocyanates, carbodiimides, isothiocyanates, glycidalethers, and acylazides.
 14. The prosthetic tissue material according to claim 1, whereinthe prosthetic tissue material is liquid-crosslinked using aqueousglutaraldehyde.
 15. The prosthetic tissue material according to claim 1,wherein the collagen-based tissue is selected from the group consistingof pericardium, dura matter, heart valves, blood vessels, fascia,ligament, tendon, and pleura tissue.
 16. The prosthetic tissue materialaccording to claim 1, wherein the collagen-based tissue is selected fromthe group consisting of bovine pericardium, porcine pericardium, andovine pericardium.
 17. The prosthetic tissue material according to claim1, wherein the collagen material is comprised of about 99% collagencontent.